The Difference in Cytotoxic Activity Between Two Optical Isomers Of

molecules

Communication The Diﬀerence in Cytotoxic Activity between Two Optical Isomers of Gelsemine from Gelsemium elegans Benth. on PC12 Cells

Li Lin 1, Yan-Chun Liu 1,2 and Zhao-Ying Liu 1,2,*

1 College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China; [email protected] (L.L.); [email protected] (Y.-C.L.) 2 Hunan Engineering Technology Research Center of Veterinary Drugs, Hunan Agricultural University, Changsha 410128, China * Correspondence: [email protected]; Tel.: +86-151-1117-9464

Academic Editors: Francesco Epifano and Serena Fiorito Received: 2 May 2019; Accepted: 22 May 2019; Published: 25 May 2019

Abstract: Two optical isomers, +/ gelsemine (1, 2), together with one known compound were isolated − from the whole plant of G. elegans. The structures of the separated constituents were elucidated on 1D and 2D (1H-1H COSY, HMBC, HSQC) NMR spectroscopy and high-resolution mass spectrometry (HRMS). The isolated alkaloids were tested in vitro for cytotoxic potential against PC12 cells by the MTT assay. As a result, (+) gelsemine (compound 1) exhibited cytotoxic activity against PC12 cells with an IC value of 31.59 µM, while ( ) gelsemine (compound 2) was not cytotoxic. 50 − Keywords: Gelsemium elegans; gelsemine; koumine; PC12 cells

1. Introduction Gelsemium elegans Benth. (G. elegans), referred to as “Gou-Wen” and “Duan-Chang-Cao” in China, belongs to Loganiaceae family and is a famous toxic plant that is widely distributed in Southeast Asia and southern China [1]. This plant has been used as a traditional medicine for the treatment of pain, spasticity, skin ulcers, migraines, neuralgia, sciatica, cancer, and various types of sores [2–8]. The Gelsemium genus includes three species, all of which have a large number of alkaloids, including indole, bisindole, and monoterpenoid alkaloids [7,9–11]. To date, more than 120 kinds of alkaloids, including some stereo-isomer compounds, have been isolated from G. elegans. Most of the alkaloids are classified into five types based on their chemical structure characteristics: gelsedine-, koumine-, humantenine-, sarpagine-, and gelsemine-type [5,12]. The Gelsemium alkaloids have potent cytotoxic, analgestic, anti-inflammatory, immunomodulating, and antiarrhythmic activities, which means they have potential as new drugs [7,13]. Their tremendous effects and the unusual and densely functionalized nature of the hexacyclic structure have been used in the development of synthetic approaches toward gelsemine, humantenine, and gelsedine-type alkaloids. The first total syntheses of ( )-gelsemine were ± disclosed in 1994 by the groups of Johnson. Gelsemine and koumine of G. elegans inhibited Tetrahymena thermophila cells’ growth in a dose-dependent manner [14,15]. In the present study, two optical isomers, +/ gelsemine (1, 2), together with one known compound, koumine, were isolated from the whole − plant of G. elegans (Figure1). According to the structure of gelsemine, gelsemine has seven chiral carbon and two chiral nitrogen atoms. The structures of these compounds were elucidated mainly by NMR spectroscopic and high resolution mass spectroscopic methods. Furthermore, all compounds have been tested in vitro for cytotoxic potential against PC12 cells by MTT assay.

Molecules 2019, 24, 2004; doi:10.3390/molecules24102004 www.mdpi.com/journal/molecules Molecules 2019, 24, x 2 of 6 Molecules 2019, 24, 2004 2 of 6 2. Results and Discussion Molecules 2019, 24, x 2 of 6

2. Results and Discussion

Figure 1. The structures of compound ((±)-gelsemine)-gelsemine ( 1//2) and koumine ( 3). ±

2. Results and Discussion 1 13 Compound 1 wasFigure isolated 1. The structuresas a white of powdercompound (for (±)-gelsemine H and C-NMR,(1/2) and koumine see Table (3). 1). The molecular formulaCompound was determined1 was isolated as C20H as23N a white2O2 bypowder HR-ESI-MS (for 1inH positive and 13C-NMR, mode (m/z see 323.1758 Table1). The[M + molecular H]+, calcd + 1 13 1 13 + formulafor [M +Compound wasH] 323.1751). determined 1 was The isolated as H C 20andH as23 NaC-NMR white2O2 by powder (See HR-ESI-MS supplementary (for H inand positive C-NMR, material mode sees (FigureTablem/z 323.1758 1). S1–S6) The molecular [Mprofile+ H] of , calcdcompoundformula for [M 1was +matchedH] determined+ 323.1751). with asthe C Thereported20H231NH2O and 2data by 13HR-ESI-MS forC-NMR gelsemine (See in positive supplementary[16]. mode (m/z materials 323.1758 [M Figures + H]+, S1–S6)calcd + 1 13 proﬁleforCompound [M ofcompound + H] 323.1751). 2 was1 matchedisolated The H as with and a yellow theC-NMR reported powder (See data supplementaryand for had gelsemine similar material NMR [16]. datas Figure to 1 S1–S6)(Table profile1), but ofthe puritycompoundCompound of compound 1 matched2 was 2 isolatedwas with lower the as reported athan yellow 1. Thedata powder molecularfor gelsemine and had formula [16]. similar was NMR determined data to 1 as(Table C20H123),N but2O2 the by Compound 2 was isolated as a yellow powder+ and had similar NMR+ data to 1 (Table 1), but the purityHR-ESI-MS of compound in positive2 was mode lower (m/z than 323.17631. The [M molecular + H] , calcd formula for [M was + H] determined 323.1751). asThe C 20retentionH23N2O time2 by purity of compound 2 was lower than 1. The molecular formula was determined2 as C20H23N2O2 by HR-ESI-MSof compound in 1 positive and 2 was mode 16.861 (m/z min323.1763 and 16.619 [M + H] min,+, calcd respectively. for [M + H]The+ 323.1751).MS spectra The of retentioncompounds time 1 HR-ESI-MS in positive mode (m/z 323.1763 [M + H]+, calcd for [M + H]+ 323.1751). The retention time ofand compound 2 are shown1 and in Figure2 was 16.8612. The minproduct and 16.619ions of min, compounds respectively. 1 and The 2 were MS2 quitespectra similar. of compounds According1 of compound 1 and 2 was 16.861 min and 16.619 min, respectively. The MS2 spectra of compounds 1 andto the2 are data shown of 1H in and Figure 13C-NMR(See2. The product supplementary ions of compounds material1 ands Figure2 were S7–S12), quite similar. compound According 2 was to and 2 are shown in Figure 2. The product ions of compounds 1 and 2 were quite similar. According theidentified data of as1H gelsemine. and 13C-NMR(See supplementary materials Figures S7–S12), compound 2 was identiﬁed to the data of 1H and 13C-NMR(See supplementary materials Figure S7–S12), compound 2 was as gelsemine.identified as gelsemine.

(a) (a)

(b(b) ) Figure 2. The MS/MS spectra of compound 1 (a) and 2 (b). FigureFigure 2. 2.The The MS MS/MS/MS spectra spectra of of compound compound 11 ((aa)) andand 2 (b).

Molecules 2019, 24, 2004 3 of 6

Compound 3 was isolated as a yellow powder with the following properties: positive ESI-MS m/z: 307.1812 [M + H]+, 13C-NMR (See supplementary materials Figure S13) (101 MHz, MeOD) δ 187.85 (2-C), 155.04 (13-C), 144.59 (8-C), 137.95 (19-C), 129.51 (11-C), 127.72 (10-C), 124.58 (9-C), 121.47 (12-C), 116.93 (18-C), 71.88 (3-C), 61.91 (17-C), 58.97 (7-C), 58.15 (21-C), 57.77 (5-C), 46.59 (20-C), 42.84 (N-Me), 38.52 (16-C), 33.97 (15-C), 30.46 (6-C), 25.93 (14-C). 1H-NMR(See supplementary materials Figure S14) (400 MHz, MeOD) δ 7.65 (1H, d, J = 7.3 Hz, 12-H), 7.55 (1H, d, J = 7.6 Hz, 9-H), 7.40 (1H, td, J = 7.6, 1.2 Hz, 11-H), 7.33 (1H, td, J = 7.5, 1.0 Hz, 10-H), 4.92 (1H, dd, J = 8.3, 1.7 Hz, 18α-H), 4.82 (dd, J = 11.2, 0.8 Hz, 1H, 18β-H), 4.69 (1H, dd, J = 17.6, 11.3 Hz, 19-H), 4.32 (1H, dd, J = 12.0, 4.6 Hz, 17α-H), 3.64 (1H, d, J = 12.0 Hz, 17β-H), 3.29 (1H, d, J = 11.9 Hz, 21α-H), 3.03 (1H, m, 21β-H), 2.88 (1H, m, 5-H), 2.67 (1H, s, N-Me), 2.42 (2H, m, 6-H), 1.87 (dt, J = 14.8, 2.0 Hz, 1H, 14β-H). This proﬁle was consistent with the reported NMR data for koumine [16].

Table 1. 1H-NMR (400 MHz) and 13C-NMR (100 MHz) data for Compound 1 and 2 in MeOD (δ in ppm and J in Hz).

Compound 1 Compound 2 Position δC δH δC δH 2 179.26 / 180.56 / 3 69.58 3.60 (1H, s) 70.97 3.64 (1H, s) 5 71.81 4.12 (1H, d, 11.0) 73.28 4.13 (1H, d, 11.0) 6 50.63 1.99 (1H, s) 51.93 2.01 (1H, s) 7 54.19 / 55.55 / 8 131.70 / 133.00 / 9 127.99 7.46 (1H, d, 7.6) 129.30 7.46 (1H, d, 7.6) 10 121.23 7.00 (1H, t, 7.6) 122.65 7.00 (1H, t, 7.6) 11 128.10 7.22 (1H, t, 7.7) 129.49 7.22 (1H, t, 7.7) 12 108.97 6.87 (1H, d, 7.7) 110.40 6.87 (1H, d, 7.7) 13 141.20 / 142.60 / 2.04 (1H, m) 2.04 (1H, m) 14 22.34 23.70 2.84 (1H, dd, 14.3, 2.1) 2.84 (1H, m) 15 35.63 2.34 (1H, t) 36.98 2.38 (1H, s) 16 37.50 2.49 (1H, m) 38.97 2.50 (1H, m) 3.97 (1H, d, 11.0) 3.97 (1H, m) 17 60.81 62.14 3.72 (1H, s) 3.72 (1H, s) 4.98 (1H, d, 17.8) 4.98 (1H, d, 17.8) 18 111.59 113.16 5.05 (1H, d, 11.0) 5.06 (1H, d, 11.0) 6.25 (1H, dd,17.8, 19 138.31 6.26 (1H, dd, 17.8,11.0) 139.48 11.0) 20 53.68 / 55.02 / 2.78 (1H, d,10.8) 2.80 (1H, d, 10.7) 21 65.17 66.45 3.33 (1H, d, 1.3) / N-CH3 39.03 2.28 (3H,s) 40.43 2.31 (3H, s)

The effects of the three compounds on the activity of poorly differentiated PC-12 cells and highly differentiated PC-12 cells (Figure3) were investigated, and it was found that only compound 1 inhibited viability in highly differentiated PC-12 cells, while the other compounds were not toxic to either type of PC-12 cells. As shown in Table2, compound 1 on poorly differentiated PC-12 cells showed no toxicity (IC50 > 100 µM), and did not affect its activity. However, compound 1 showed toxicity to highly differentiated PC-12 cells and decreased cell activity, and its IC50 was 31.59 µM. The results indicated that compound 1 exhibited some cytotoxic activity against PC12 cells, while compound 2 did not. 2D (1H-1H COSY, HMBC, HSQC) NMR spectroscopy (Figures S1–S12) showed that compounds 1 and 2 are quite similar. However, the optical rotation results showed that compound 1 and 2 are two optical isomers of gelsemine (1: α D = 793.79 (c = 0.047, MeOH), 2: α D = 909.09 (c = 0.025, [ ]25 [ ]25 − MeOH)) which explained why they differ in cytotoxic activity on PC12 cells. Molecules 2019, 24, 2004 4 of 6 Molecules 2019, 24, x 4 of 6

150 Compound 1 120 a Compound 2 b 120 Compound 3 90 90 60 60 30 30 Cell Viability (% Control) 0 Cell Viability (% Control) 0 10 0 10 1 102 103 10 -3 10-2 10 -1 100 10 1 10 2 conc. [μM] conc. [μM]

c d 150 120 Compound 1 120 Compound 2 90 Compound 3 90 60 60 30 30 Cell Viability (% Control) Cell Viability (% Control) 0 0 10 0 101 102 10 3 10 -3 10 -2 10 -1 10 0 10 1 10 2 conc. [μM] conc. [μM]

Figure 3. The cell viability of the three compounds in a series of concentrations (5, 10, 25, 50, 100 µM) Figure 3. The cell viability of the three compounds in a series of concentrations (5, 10, 25, 50, 100 μM) and positive drug staurosporine (STSP) on two types of PC-12 cells after exposure for 48 h. (a) the and positive drug staurosporine (STSP) on two types of PC-12 cells after exposure for 48 h. (a) the three compounds’ concentration responses of poorly differentiated PC-12 cells; (b) STSP concentration three compounds’ concentration responses of poorly differentiated PC-12 cells; (b) STSP response in poorly differentiated PC-12 cells; (c) the three compounds’ concentration responses in concentration response in poorly differentiated PC-12 cells; (c) the three compounds’ concentration highly differentiated PC-12 cells; (d) STSP concentration response in highly differentiated PC-12 cells. responses in highly differentiated PC-12 cells; (d) STSP concentration response in highly

differentiatedTable PC-12 2. cells.Cytotoxicity of compounds 1–3 against PC12 cell lines (IC50, µM)

CompoundTable 2. Cytotoxicity PC12 Highly of compounds Diﬀerentiated 1–3 against Cells PC12 PC12 cell Poorly lines (IC Di50ﬀ, erentiatedμM) Cells 1 >100 31.59 Compound PC12 Highly Differentiated Cells PC12 Poorly Differentiated Cells 2 >100 >100 1 >100 31.59 3 >100 >100 2 >100 >100 STSP 0.1944 0.022 3 >100 >100 STSP 0.1944 0.022 3. Materials and Methods 3. Materials and Methods 3.1. General Experimental Procedures 3.1. NMRGeneral spectra Experimental were acquiredProcedures using a Bruker ACF-400 spectrometer (the 1H-NMR spectra at 400 MHzNMR and spectra13C-NMR were spectra acquired at 100using MHz, a Bruker (Bruker, ACF-400 Rheinstetten, spectrometer Germany). (the The1H-NMR mass spectra wereat 400 determinedMHz and 13 byC-NMR an Agilent spectra 1290 at HPLC100 MHz, system (Bruker, (Agilent Rheinstetten, Technologies, Germany). California, The CA, mass USA) spectra coupled were withdetermined a 6530 Q-TOF by an/ MSAgilent accurate-mass 1290 HPLC spectrometry. system (Agilent Silica Technologies, gel (100–200, California, 200–300, 300–400 CA, USA) mesh) coupled was usedwith for a 6530 column Q-TOF/MS chromatography accurate-mass (CC) spectrometry. and silica GF254 Silica for gel TLC (100–200, was supplied 200–300, by 300–400 Qingdao mesh) Haiyang was Chemicalused for column Factory, chromatography Qingdao, RP China. (CC) All and solvents silica GF254 used were for TLC of analytical was supplied grade by and Qingdao obtained Haiyang from ShanghaiChemical Chemical Factory, Qingdao, Reagents RP Company, China. All Shanghai, solvents RPused China. were of Acetonitrile analytical grade and methanol and obtained used from for chromatographicShanghai Chemical grade Reagents were purchased Company, from Shanghai, Merck, German.RP China. PC12 Acetonitrile cells were and purchased methanol from used Cell for bankchromatographic of Chinese academy grade were of sciences. purchased Staurosporine from Merck, (STSP) German. was PC12 obtained cells fromwere MedChempurchased from Express, Cell Shanghai,bank of Chinese China. academy of sciences. Staurosporine (STSP) was obtained from MedChem Express, Shanghai, China. 3.2. Plant Material 3.2. ThePlant whole Material plant of G. elegans was collected from Longyan city, Fujian province (24.699739 N, 116.98967The E).whole The plant samples of G. were elegans authenticated was collected by Dr from Qi Tang Longyan at Hunan city, AgriculturalFujian province University. (24.699739 N, 116.98967 E). The samples were authenticated by Dr Qi Tang at Hunan Agricultural University.

Molecules 2019, 24, 2004 5 of 6

3.3. Extraction and Isolation The air-dried whole plant of G. elegans (100 kg) was powdered and extracted two times (2 24 h) × with 0.5 % sulfuric acid solution (100 L) at room temperature. The extracted solution was filtered through a 200-mesh filter and combined, then was basified with NaOH (8 mol/L) to pH 7.0, and dried under reduced pressure. Then, the fraction (1 kg) was extracted by reflux in 1.5 L of 95 % ethanol for twice times (each for 2 h). After filtration, the extract was combined and concentrated under reduced pressure. The ethanol fraction (138 g) was further fractionated through a silica gel column chromatography (CC), eluting with CH2Cl2–MeOH (from 100:0 to 0:100, v/v) to obtain 14 fractions according to TLC analysis (Frs. 1–14). Fraction 7 (2.85 g) was subjected to silica gel CC (CH2Cl2-MeOH -ammonia, 95:5:0.05, v/v/v) to afford compound 1 (78 mg). Fraction 3 (5.00 g) was subjected to silica gel CC (CH2Cl2-MeOH-ammonia, 95:5:0.05–90:10:0.05, v/v/v) to afford three subfractions (Fr. 3-1 and Fr. 3-3). Fr. 3-1 (0.50 g) was subjected to silica gel CC (CH2Cl2–MeOH-ammonia, 20:0.8:0.05, v/v/v) to obtain compound 2 (71 mg) and compound 3 (99 mg).

3.4. Cell Line and Culture Poorly differentiated PC-12 cells: PC12 cells (Cell bank of Chinese academy of sciences) were grown in 100 cm2 culture flasks (1 106 cells per flask) at 37 C in serum-containing medium, 85% RPMI × ◦ 1640, 10% heat-inactivated horse serum (Gibco, Grand Island, NY, USA ), 5% fetal calf serum (Biowest, Nuaillé, France ), penicillin & streptomycin (1 P/S, Procell, Tongxiang, China, Lot: 16050130) and no × NGF. Culture medium was exchanged three times weekly. Highly differentiated PC-12 cells: PC-12 cells were grown in serum-containing medium (90% RPMI 1640, 10% fetal calf serum, 1 P/S), treated × with 20 ng/mL of NGF (Sigma, San Francisco, CA, USA, Lot: SRP3018). The medium was changed every 2–3 days and NGF was added if required.

Supplementary Materials: The following are available online, Figure S1: The 13C-NMR spectrum of compound 1, Figure S2. The 1H-NMR spectrum of compound 1, Figure S3: The 1H-1H COSY spectrum of compound 1, Figure S4: The HMBC spectrum of compound 1, Figure S5: The HSQC spectrum of compound 1, Figure S6: The DEPT135 spectrum of compound 1, Figure S7: The 13C-NMR spectrum of compound 2, Figure S8: The 1H-NMR spectrum of compound 2, Figure S9: The 1H-1H COSY spectrum of compound 2, Figure S10: The HMBC spectrum of compound 2, Figure S11: The HSQC spectrum of compound 2, Figure S12: The DEPT135 spectrum of compound 2, Figure S13: The 13C-NMR spectrum of compound 3, Figure S14: The 1H-NMR spectrum of compound 3. Author Contributions: Z.-Y.L. conceived and designed the experiments; Y.-C.L. performed extraction, isolation, MS experiment; L.L. performed NMR experiments and contributed to the preparation of the manuscript; Z.-Y.L. revised the manuscript. Funding: Support was received from the Hunan Provincial Natural Science Foundation of China (Grant No. 2017JJ1017) Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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